Over 10% of the US population suffers from gastrointestinal (Gl) tract disorders. Reciprocal signaling among Gl tract components, including the gut epithelium, enteric nervous system (ENS), and gut microbiota are crucial for Gl tract health and homeostasis. Disruption of this reciprocal signaling can result in diseases such as inflammatory bowel disease (IBD). We and others have shown that the resident microbiota are crucial for normal gut development. We have also identified a previously unknown role for gut microbiota in establishing the normal cellular composition of both the developing gut epithelium and the developing ENS: in the absence of gut microbiota, there are fewer gut epithelium secretory cells and more ENS glia. These developmental defects are exactly opposite to developmental defects seen in Notch pathway mutants. Our observations motivate the hypothesis that gut microbiota influence developmental cell fate decisions in the gut epithelium and ENS by inhibiting Notch signaling. We propose 3 Aims to address this hypothesis: 1) We will use genetic mosaics to test whether Notch signaling is required autonomously in the gut epithelium and the ENS to specify cell fate decisions in each of these tissues. 2) We will manipulate host gene function and microbial associations to test whether the microbiota affect cell fates in the gut epithelium and ENS by repressing Notch signaling. 3) We will identify genes that specify ENS and gut epithelium cell fates and test whether they function in microbially-regulated pathways. These experiments will reveal the mechanisms by which Notch signaling affects developmental cell fate decisions in the gut epithelium and ENS and identify which steps in the pathway are affected by the microbiota. Because the gut epithelium is in a state of constant renewal, learning the nature of these developmental decisions will have important ramifications for understanding cell fate decisions in the gut throughout life. Together our studies will provide a more complete understanding of the molecular mechanisms that promote gut development and health, knowledge that is urgently needed to design better diagnostics and therapies for debilitating and sometimes fatal gut diseases such as IBD.